Insulin derivatives

ABSTRACT

A physiologically acceptable insulin derivative has the terminal amino group of the B chain (B 1 , phenylalanine) protected by an acyl group or other blocking group containing up to 7 carbon atoms and the amino group of the A chain (A 1 , glycine) is either free or protected by means of an acyl or other blocking group containing no more than four carbon atoms and preferably no more than three atoms other than hydrogen. Pharmaceutical preparations may be prepared containing an effective amount of such an insulin derivative together with a physiologically acceptable diluent. An insulin derivative of reduced antigenicity is obtainable from porcine or bovine insulin by reacting the insulin with an acylating agent or other blocking reagent to acylate or otherwise block the amino group of the B 1  amino acid and optionally block the A 1  and/or B 29  amino acid, followed by purification of the derivative by various methods.

This is a division of application Ser. No. 390,216 filed Aug. 21, 1973now U.S. Pat. No. 3,869,437, which itself is a continuation-in-part ofSer. No. 89,816 filed Nov. 16, 1970, now abandoned.

This invention relates to insulin derivatives.

Porcine and bovine insulin have been used clinically for many years inthe treatment of diabetes and other disorders. One of the disadvantagesof these materials however is that for certain patients it is necessaryafter treatment has been continued for some time to increase the dose inorder to produce the required effect. This has been explained byascribing antigenic properties to those non-human insulins which giverise to antibodies in sufficient amount to counteract a proportion ofthe dose of insulin applied.

It has now been found possible to reduce considerably and perhaps eveneliminate the ability of porcine and bovine insulins to react withspecific insulin antibody without unduly affecting the desirableproperties of these hormones.

In accordance with this invention physiologically acceptablesubstantially pure mono-, di- or tri-substituted insulin derivativeshaving reduced reactivity to insulin antibodies are those in which theterminal amino group of the B chain (B₁ phenylalanine) is protected byan acyl group or other blocking group containing up to 7 carbon atomsand the amino group of the A chain (A₁, glycine) is either free orprotected by means of an acyl or other blocking group containing no morethan four carbon atoms and no free primary amino group. The blockinggroups in each case may be straight or branched chain or cyclic radicalscontaining carbon and if desired other elements including, for example,oxygen, nitrogen and sulphur. The term "pure" is used in thisspecification to mean free from by-products of manufacture. It thereforeembraces not only monocomponent preparations in which the customarypharmaceutical additives are present but multicomponent preparationsbased on individually pure and defined components. Another very valuabletype of blocking group on the A₁ glycine amino group is one whichcontains no more than three atoms other than hydrogen.

Although insulin has been subjected to many substitution reactions inthe past the results recorded in the literature are complex anddifficult to interpret due chiefly to the fact that such reactions giverise to a complex mixture of products and unless proper care is taken inthe separation and purification of various products, it is impossible toreach firm conclusions as to the biological properties of thederivatives so formed. Most workers have reported a substantial loss inactivity and it has not been recognised, prior to the present invention,that by careful selection of the substituent protective groupsderivatives may be produced which have blood sugar lowering propertiesvery similar to that of the parent insulin, at least as regards theinitial effect upon intravenous injection into experimental animals, buthave significantly reduced reactivity towards specific insulinantibodies. To achieve these effects the nature and size of theprotective group on the B chain in the compounds of the presentinvention is not as critical as that on the A chain and it will beappreciated that different blocking groups may be used for therespective amino groups. However in practice it is considerably moreconvenient to use the same blocking group for both amino groups, andalso for the third amino group of the B₂₉ amino acid (lysine) wheretri-substituted derivatives are formed.

Protection of the desired amino groups is readily achieved by acylation.Subject to the qualifications expressed above with regard to theprotective groups, a wide variety of acylating agents may be used tointroduce groups such as formyl, acetyl, trifluoroacetyl,hydroxypropionyl, cyclopropane-carbonyl, aceto-acetyl and otheraliphatic acyl groups, benzoyl and other aroyl groups as well as thosederived from heterocyclic compounds e.g.2,2-dimethyl-3-formyl-L-thiazolidine-4-carboxylic esters. Acylation maybe conducted by any of the standard methods employed in peptidechemistry including especially the use of activated esters or anhydridesand typical carbodiimide coupling reagents. Acylating agents which areesters of N-hydroxysuccinimide are particularly advantageous. A varietyof alternative blocking groups include those which introduce thecarbamyl, thiocarbamyl, alkyl carbamyl and alkyl thiocarbamyl groups,amidino groups, ##EQU1## and the guanidino group, ##EQU2## The amidinogroup ##EQU3## for example, is introduced by means of ethyl acetimidatehydrochloride and the guanidino group by means of O-methyl isourea.Other blocking groups are, for example HOCH₂ CH₂ CO-- (introduced bybutyrolactone) HOOC.CH₂ CO-- and the corresponding group H₂ NOC.CH₂ CO--(introduced by the corresponding activated ester of malonic acid ormalonamide). The use of more complex acylating and other groups mayintroduce more difficult separation problems and it is believed thatsimple acetylation will in practice be most attractive and conductingthe reaction to produce the greatest yield of the triacetyl (A₁, B₁, B₂₉substituted), derivative is a particularly recommended procedure inaccordance with this invention.

In order to produce the maximum yield of insulin acylated at the B₁amino group the proportion of acylating agent or other blocking reagentused is preferably relatively low. For example, reacting one mole of theinsulin with from about one to not more than about two moles ofacylating agent produces the B₁ mono-substituted derivative in thelargest amounts. Mono-substitution at other amino groups can howeveralso occur leading to by-products which are less useful. Furthermore,after substitution at B₁ a certain degree of di-substitution and eventri-substitution can take place especially when three to four moles ofacylating or other reagent are used. It has also been found that theacylation reaction depends on the pH of the reaction medium. To producethe best yield of mono-substitution product at the B₁ amino group, thepH is preferably at or near about 7.0 and preferably no greater thanabout 8. At pH 8.5 to 9 for example the yield of this desired productfalls off considerably in favour of additional substitution at A₁ andB₂₉. Usually it will be desirable to isolate the required derivative bymeans of chromatography, electrophoresis, or any other conventionalmethod of purifying peptides amenable to use on a large scale.

The insulin derivatives of the present invention exhibit of the order ofat least 75% and preferably at least 90% of the normal insulin type ofblood sugar lowering activity, weight for weight, as tested byintravenous injection into laboratory animals and measurement of theeffect within a 30 minute period after injection. In most cases the newderivatives have an initial bloor sugar lowering effect which isvirtually indistinguishable from insulin itself. In contrast thederivatives have less than about 50% of the immunoreactivity of theparent insulin as determined by radioimmunoassay. In many cases thereduction in immunoreactivity (reactivity towards specific insulinantibody) is as low as 30% or even 10% of that of the parent hormone.Comparative figures are shown in the following Tables. The figures areexpressed on a percentage basis relative to insulin.

                                      TABLE 1                                     __________________________________________________________________________                        Mouse convulsion assay                                                                    Radioimmunoassay                                                  Mean potency                                                                              Immunoreactivity                              __________________________________________________________________________    Crystalline bovine insulin                                                                        100         100                                           Phe.sup.B1 -monosubstituted insulins                                           (a) Acetyl (bovine)                                                                              100         30                                             (b) Acetoacetyl (bovine)                                                                         109         8.6                                            (c) Thiazolidine (bovine)                                                                        103         27                                            Gly.sup.A1 -monosubstituted insulins                                           (a) Acetyl (bovine)                                                                              105         71                                             (b) Acetoacetyl (bovine)                                                                         88          87                                             (c) Thiazolidine (bovine)                                                                        57          81                                            Lys.sup.B29 -monosubstituted insulins                                          (a) Acetyl (bovine)                                                                              114         83                                             (b) Thiazolidine (bovine)                                                                        101         84                                            Phe.sup.B1, Gly.sup.A1 -disubstituted insulins                                 (a) Acetyl (porcine)                                                                             90          25                                             (b) Acetoacetyl (bovine)                                                                         76          14                                            Gly.sup.A1, LYs.sup.B29 -disubstituted insulin                                 (a) Acetyl (porcine)                                                                             93          67                                            Phe.sup.B1, Gly.sup.A1, Lys.sup.B29 -trisubstituted                           insulins                                                                       (a) Acetyl (porcine)                                                                             96          10                                             (b) Acetoacetyl (bovine)                                                                         77          17                                            __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________                        Blood sugar lowering                                                                      Immunoreactivity                              __________________________________________________________________________    Crystalline bovine insulin                                                                        100         100                                           Phe.sup.B1 -monosubstituted insulin                                            (a) carbamyl       100         23                                             (b) methylthiocarbamyl                                                                           100         28                                            Gly.sup.A1 -monosubstituted insulin                                            (a) carbamyl       100                                                        (b) methylthiocarbamyl                                                                           100         92                                            Phe.sup.B1 -Gly.sup.A1 -disubstituted insulins                                 (a) carbamyl       100         10                                             (b) methylthiocarbamyl                                                                           100         20                                            Phe.sup.B1 -Gly.sup.A1 -Lys.sup.B29 tricarbamyl                               insulin             100         9                                             __________________________________________________________________________

Insulin derivatives in accordance with this invention may be formulatedas pharmaceutical preparations in the same way as the parent insulinsand may be used clinically at comparable and even lower dosage levels.Thus the desired derivative, after fractionation of the reaction mixtureand removal of excess salts e.g. by dialysis, may be recovered insolution form or solid form e.g. by freeze-drying following which it maybe made up to the required concentration in an injectablephysiologically acceptable diluent such as sterile pyrogen-free water orsaline containing suitable buffers, and dispensed in ampoules.

Examples of typical derivatives produced in accordance with theinvention will now be described.

EXAMPLE 1 a. N-Hydroxysuccinimide ester of2,2-dimethyl-3-formyl-L-thiazolidine-4-carboxylic acid

3-Formyl-2,2-dimethyl-L-thiazolidine-4-carboxylic acid (9.46 g., 0.05moles) was dissolved in redistilled dimethylformamide (50 ml.) and thesolution cooled to 4°. Recrystallised N-hydroxy-succinimide (5.76 g.,0.05 moles) was added followed by N,N'-dicyclohexylcarbodiimide (10.32g., 0.05 moles) and the solution stirred overnight at 4°, and for afurther 2 hours at 25°. The dicyclohexylurea formed was filtered off andthe dimethylformamide removed on a rotary evaporator under reducedpressure. The additional precipitate of dicyclohexylurea produced wasfiltered off and the urea was washed with dichloromethane. Petroleumether (60°-80°) was added to the supernatant until the solution wentturbid. Filtration of the crystalline material produced on standingafter drying in a desiccator containing sodium hydroxide and paraffinwax, gave colourless needles of the N-hydroxysuccinimide derivative(13.7 g., 95.7%) m.p. 98°-100°.

b. Reaction of insulin with N-hydroxysuccinimide ester of2,2-dimethyl-3-formyl-L-thioazolidine-4-carboxylic acid

Zinc insulin (500 mg., 0.085 mM) (porcine) 10 times recrystallised (NovoIndustri A/S, Copenhagen) was dissolved in hydrochloric acid (45 ml.,0.1N) and the pH adjusted with N sodium hydroxide to 6.9 in thetitration vessel of a Radiometer (Type TTT1) pH-stat. Thehydroxysuccinimide ester (22 mg., 0.085 mM) was dissolved in dioxane(100μ1) and added to the above solution in 10μ1 aliquots, maintainingthe pH at 6.9 by the addition of alkali, every 30 minutes.

After the final addition of the hydroxysuccinic ester the reactionmixture was made 0.2 M in sodium carbonate-sodium bicarbonate buffer pH9.5 and left overnight. The material was lyophilised, after extensivedialysis against distilled water, to yield 490 mg. of the modifiedprotein.

In a second experiment, zinc insulin (500 mg. 0.085 mM) was treated withhydroxysuccinic ester (32 mg., 0.127 mM) at pH 8.5 exactly as describedabove. The reaction rate as judged by the uptake of alkali was muchfaster than at pH 6.9 and aliquots were added every 5 minutes.

c. Separation of thiazolidine insulins

i. Chromatography

The thiazolidine insulins were separated on a column of DEAE-SephadexA-25 (2.5 × 40 cm.).

The column was equilibrated with buffer containing 0.01 M tris and 0.05M sodium chloride in 7 M de-ionised urea at pH 7.29. Thiazolidinemodified insulin was dissolved in this buffer (500 mg., 50 mg./ml.) andthe column developed at a flow rate of 54.0 ml./hour collecting 12 ml.fractions. After 120 ml. of eluant was collected, a linear gradient,obtained by running 0.01 M tris, 0.01 sodium chloride in 7 M de-ionisedurea (1 l.), at pH 7.29 into the stirred reservoir of the startingbuffer, (1 l.) was applied.

The protein concentration was determined from the absorbance of thesolution at 277 mμ.

The appropriate fractions were pooled and extensively dialysed againstdistilled water. The material was lyophilised and the residue de-ionisedon a column (1.5 × 60 cm.) of G.10 Sephadex equilibrated with 5% aceticacid. The protein fractions were collected and lyophilised.

The chromatographic separation of the insulin derivatives formed fromthe above reaction can be seen in FIG. 1. Using a gentle salt gradientof 0.05 to 0.1 molar sodium chloride, incomplete separation of the peaksA, B and C occured. Material under peaks A, B and C was combined andre-chromatographed under exactly the same conditions to give the elutionpattern shown in FIG. 2. Material about the centre of each peak waspooled and each individual peak subjected to rechromatography to givethree chromatographically homogenous fractions. The derivativescorresponding to peaks A, B and C were shown to be mono-substituted onphenylalanine, glycine and lysine respectively.

The elution pattern of the derivatives obtained when insulin was treatedwith a 1.5 molar excess of the acylating agent at pH 8.5 can be seen inFIG. 3. A much simpler elution pattern was obtained and materialcorresponding to derivative A in the previous chromatography was presentin low amount.

ii. Cellulose acetate electrophoresis

For the separation of the thiazolidine insulins, the conditions werethose described by Carpenter and Hayes (Biochemistry 2 (1963) 1272))using a Shandon electrophoresis tank fitted with a water-cooled plate(20 × 20 cm.) and a power supply of 1 Kv. The buffer used was 0.065 Mphosphate, 7 M urea, pH 6.5 and the cellulose acetate strips (20 × 5cm.) were stained in a 0.2% solution of Ponceau S in 3% acetic acid.

Cellulose acetate electrophoresis showed derivatives A, B and C to beelectrophoretically pure components which were mono-substituted bycomparison with unsubstituted insulin and diacetoacetyl insulin preparedpreviously.

EXAMPLE 2 Reaction Of Insulin With Diketen

Zinc insulin (1.0 g.) as in Example 1, was dissolved to a finalconcentration of 0.167 mM in 0.1 M-HCl (45 ml.) and the pH adjusted withM-NaOH to 6.9 in the titration vessel of a Radiometer (type TTT1)pH-stat. Freshly distilled diketen was added in 5μ portions, the pHbeing maintained at 6.9 by the addition of alkali. When 1 equiv. ofalkali was consumed, after approximately 5 minutes a sample was removedfor ninhydrin analysis (Moore & Stein, 1954). Further portions ofdiketen were added until the ninhydrin colour yield had decreased by30%. The amount of diketen added was 40μ1 (final concentration 0.49 mM).

The reaction mixture was made 0.2 M in Na₂ CO₃ --NaHCO₃ buffer, pH 9.5and left overnight. After extensive dialysis against distilled water andfreeze-drying, the yield of modified protein was 950 mg.

Chromatographic Separation of Acetoacetyl Insulins

The acetoacetyl insulins were separated on a column of DEAE-SephadexA.25 (2.5 cm. × 40 cm.).

The column was equlibrated with buffer containing 0.01 M-tris and 0.05M-NaCl in 7 M de-ionised urea adjusted to pH 7.20 with M-HCl.Acetoacetyl insulin (500 mg.) was dissolved in this buffer (50 mg./ml.)and the column was developed at a flow rate of 54.0 ml./hr., 12 ml.fractions being collected. After 120 ml. of eluant was collected alinear gradient, obtained by running 0.01 M-tris- 0.15 M-NaCl in 7 Mde-ionised urea at pH 7.20 into the stirred reservoir of the startingbuffer was applied.

The mono-acetoacetylated insulins were rechromatographed on a column ofDEAE-Sephadex A-25 (2.5 cm. × 40 cm.) as described above with theexception that the starting buffer was adjusted to pH 7.30 with M-HCl. Alinear gradient was applied by running 0.01 M-tris- 0.10 M-NaCl in 7Mde-ionised urea at pH 7.30 into the stirred reservoir of the startingbuffer.

The protein concentration was determined from the extinction of thesolution at 277 nm.

Chromatography results for insulin (500 mg.) treated with a threefoldmolar excess of diketen can be seen in FIG. 4. Four main peaks arediscernible. The position of elution of unmodified insulin is shown onthe Figure. Component I was mainly unmodified insulin; component IV (80mg.) was chromatographically homogeneous, requiring a higher saltconcentration for elution, and would be expected to be more highlysubstituted. No further protein was eluted.

EXAMPLE 3 N-Hydroxysuccinimide Acetate

Acetic acid (3.0 g. 0.05 mole) was dissolved in dichloromethane (30 ml.)and a solution of N-hydroxysuccinimide (5.76 g., 0.05 mole) in anhydrousdioxan (10 ml.) added. N,N'-dicyclohexylcarbodiimide (10.32 g., 0.05mole) was added and the reaction mixture was stirred overnight at 4° andfor a further 2 hours at room temperature. The dicyclohexylurea formedwas filtered off and the solvent removed on a rotary evaporator underreduced pressure. Petroleum ether (60°-80°) was added to the supernatantuntil the solution went turbid. Filtration of the crystalline materialand recrystallisation from ethyl acetate gave colourless needles ofN-hydroxysuccinimide acetate (7.5 g., 95.5%) m.p. 131°-4°.

Reaction of Insulin With N-hyroxysuccinimide Acetate

Zinc insulin (200 mg., 0.038 mmol.) was dissolved in hydrochloric acid(40 ml., 0.1 M) and the pH adjusted with sodium hydroxide to 6.9 in thetitration vessel of a Radiometer (Type TTT1) pH-stat.N-hydroxysuccinimide acetate (6 mg., 0.038 mmol.) was dissolved indioxane (100μ1) and added to the above solution in 10μ1 aliquotsmaintaining the pH at 6.9 by the addition of alkali every 30 minutes.

The reaction mixture was left overnight and was then dialysed againstdistilled water and lyophilised to yield 185 mg. of the modifiedprotein.

In a second experiment, zinc insulin (500 mg. 0.085 mmol.) was treatedwith N-hydroxysuccinimide acetate (15.2 mg. 0.085 mmol.) at pH 8.5exactly as described above, adding aliquots of the activated ester every5 minutes.

In a further experiment, zinc insulin (500 mg., 0.085 mmol.) was treatedwith the activated ester (40 mg. 0.225 mmol.) at pH 8.5 as above.

Chromatographic Separation of the Acetyl Insulins

The acetyl insulins were separated on a column of DEAE-Sephadex A-25(2.5 × 40 cm.) by a modification of the method described by Bromer &Chance (1967). This column was equilibrated with buffer containing0.01M-tris and 0.05 M-NaCl in 7 M de-ionised urea adjusted to pH 7.30with M-HCl. The acetyl insulins were dissolved in this buffer (50mg./ml.) and the column was developed at a flow rate of 54.0 ml./hr.,9.7 ml. fractions being collected. 97 ml. of eluant was collected beforea linear gradient was applied. For insulin treated with a molarequivalent of N-hydroxysuccinimide acetate, a linear gradient obtainedby running 0.01 M-tris and 0.10 M-NaCl in 7 M de-ionised urea (1 l.) atpH 7.30 into the stirred reservoir of the starting buffer (1 l.) wasapplied.

For insulins treated with a threefold molar excess ofN-hydroxysuccinimide acetate, the concentration of NaCl in the finalbuffer was increased to 0.15 M.

The protein concentration was determined from the extinction of thesolution at 277 nm.

Tubes around the centre of each peak from the chromatography were pooledand separately rechromatographed under the same conditions. This processwas repeated until each peak appeared homogeneous by iso-electricfocussing. The material was lyophilised and the residue de-ionised on acolumn (1.5 × 60 cm.) of Sephadex G.10 equilibrated with 5% acetic acid.The fractions were pooled and the protein lyophilised.

At low reagent concentrations and near neutral pH, the predominantproducts of the reaction are the two mono-substituted acetyl insulins inwhich the two terminal α-amino groups are modified (FIG. 5). However, ifthe pH is raised the amount of the Phe^(B1) acetyl insulin isolated isreduced and Lys^(B29) acetyl insulin is also produced (FIG. 6).

The chromatographic separation of the products of the reaction ofinsulin with a threefold excess of the activated ester at pH 8.5 can beseen in FIG. 7. Characterisation of the two di-substituted acetylinsulins formed shows that they are Phe^(B1) Gly^(A1) -diacetyl insulinand Gly^(A1) Lys^(B29) -diacetyl insulin respectively.

Biological Assay

The insulin derivatives were separated from trace amounts of urea andsalt by elution from a Sephadex G.10 column (2.5 × 60 cm.) equilibratedwith 5% aqueous acid and the protein, after lyophilisation, was driedover phosphorous pentoxide at room temperature for 2 days. Thederivatives were assayed against a neutral insulin solution (Nusoinsulin batch A 354) as standard by the Mouse Convulsion method (BritishPharmacopoeia, 1968). All injections were of solutions in acetatebuffer, pH 8.0.

EXAMPLE 4

The procedures described in Examples 1, 2 and 3 were repeated usingbovine insulin in place of porcine insulin. Virtually identical resultswere obtained in all cases.

EXAMPLE 5

Following the procedures described in Examples 1(b) and 1(c), themono-carbamyl, mono-methylcarbamyl and monomethylthiocarbamylderivatives of bovine insulin were prepared by reacting zinc insulin(600 mg., 100μ mole) in 100 mM phosphate buffer pH 7.5 (40 ml.) at 37°C.overnight with potassium cyanate and methyl isocyanate and methylisothiocyanate (150μ moles) respectively and subsequently extensivelydialysed and then separated by chromatography as described above. Thedesired substituted derivatives of the B₁ amino acid and other residueswere isolated and dialysed to remove excess salts and freeze dried.

EXAMPLE 6

Following the procedure of Examples 1(b) and 1(c), insulin was reactedwith methyl acetimidate hydrochloride and the resulting products wereworked up as described previously.

EXAMPLE 7 Preparation of A₁, B₁, B₂₉ -triacetyl-insulin

Insulin (100 mg., 16.7μ mole) is dissolved in anhydrousdimethylformamide (100 ml.) and redistilled triethylamine (0.5 ml.)added. N-hydroxysuccinimide acetate (130 mg., 0.84 mmole) is dissolvedin dimethylformamide (1.2 ml.) and is added to the stirred insulinsolution in 0.1 ml. aliquots, over a period of 60 minutes. The solutionis stirred overnight and then dialysed extensively against distilledwater and the dialysed solution lyophilised Yield of A₁, B₁, B₂₉triacetyl-insulin (90 mg.)

EXAMPLE 8 (Carbamyl-Phe)B₁ -insulin

600 mg. of amorphous Zn-free insulin is dissolved in 60 ml. of 0.1 molarphosphate buffer (pH 7.5); 0.3 ml. of 0.6N potassium cyanate is addedthereto and the mixture maintained at 30°C. for 18 hours. The reactionsolution is then dialysed against water until free of excess cyanate aswell as buffer salts and thereafter freeze-dried. Yield: 500 mg. Inorder to separate the (carbamyl-Phe)B₁ -insulin, the usual methods ofpeptide purification can be employed, such as ion exchangechromatography, for example with the use of DEAE-Sephadex in 4-7 molarurea buffer, or countercurrent distribution, e.g. in the systemn-butanol (20), methanol (5), water (20), glacial acetic acid (1). Theyield in pure (carbamyl-Phe)B₁ -insulin is 200 mg.

Paper electrophoresis:

Conditions: 24 molar aqueous formic acid/4 molar aqueous urea, pH 2,dyeing with Pauly reactant. 300μ g was applied.

The substance migrates as a uniform band; the Rf value of 0.91 (insulin:1.00) for this compound corresponds to a monosubstituted insulin.

EXAMPLE 9 (Carbamyl-Gly)A₁ -(Carbamyl-Phe)B₁ -insulin

6 g of amorphous, Zn-free insulin from cattle is dissolved in 600 ml. ofdemineralised water; the solution is adjusted to pH 7.2 with 1N NaOH andheated to 30°C. At this temperature, over a period of about 7 hours, 300ml. of 0.6N potassium cyanate is added dropwise to the stirred reactionmixture. During the same interval, the pH is gradually brought from 7.2to 5.5 by the addition of 1N acetic acid using an automatic titratingdevice. After termination of the reaction, excess cyanate is destroyedby acidification to a Ph of 2.2-2.5. The acidic solution is dialysedagainst distilled water until free of excess acid and salts andsubsequently freeze-dried. The yield is 5.3-5.5 g of (carbamyl-Gly)A₁-(carbamyl-Phe)B₁ -insulin. Minor amounts of mono- and tricarbamylinsulin can be removed by the usual methods of peptide purification,such as ion exchange chromatography, countercurrent distribution,carrier-free electropboresis, or advantageously by isoelectricprecipitation by dissolving the product in water at pH 7 andprecipitating it by acidification to pH 4; the mono- and tricarbamylinsulin impurities remain in solution and can be decanted off.

Paper electrophoresis:

The substance migrates as a uniform band. The Rf value is 0.76 (insulin:1.00) using the conditions of Example 1.

EXAMPLE 10 (Carbamyl-Gly)A₁ -(Carbamyl-Phe)B₁ -(Carbamyl-Lys)B₂₉-insulin

6 g of amorphous, Zn-free insulin is dissolved in 600 ml. of a pH 8.5buffer (e.g. tris buffer or phosphate buffer), and 300 ml. of 0.6Npotassium cyanate is gradually added dropwise thereto with stirring.After the addition is terminated, excess cyanate is destroyed byacidification to pH 2.5. The acidic solution is dialysed againstdistilled water until free of excess acid and thereafter freeze-dried.Yield: 5.5 g. Purification is generally unnecessary, but can readily beeffected according to the usual methods.

Paper electrophoresis:

Using the condition of Example 1, the substance migrates as a uniformband. The Rf value thereof is 0.56 (insulin: 1.00) and corresponds to atri-substituted insulin.

We claim:
 1. A pharmaceutical composition useful for treating diabetesin humans which comprises a hypoglycemically effective amount of amono-, di- or tri-substituted insulin in which the terminal amino groupof the B chain (B₁ phenylalanine) is protected by an acyl or otherblocking substituent containing up to 7 carbon atoms selected from thegroup consisting of formyl, acetyl, trifluoroacetyl,cyclopropane-carbonyl, aceto-acetyl, benzoyl,2,2-dimethyl-3-formyl-L-thiazolidine-4-carbonyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU4## HOCH₂ CH₂CO--, HOOCCH₂ CO-- and H₂ NOCCH₂ CO--, the terminal amino group of the Achain (A₁ glycine) is either free or protected by an acyl or otherblocking substituent containing no more than 4 carbon atoms and no freeprimary amino group selected from the group consisting of formyl,acetyl, trifluoroacetyl, cyclopropane-carbonyl, acetoacetyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU5## HOCH₂ CH₂CO--, HOOCCH₂ CO-- and H₂ NOC--CH₂ CO-- and the amino group of the B₂₉amino acid (lysine) is either free or protected by an acyl or otherblocking substituent containing no more than 4 carbon atoms and no freeprimary amino group selected from the group consisting of formyl,acetyl, trifluoroacetyl, cyclopropanecarbonyl, acetoacetyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU6## HOCH₂ CH₂CO--, HOOC--CH₂ --CO-- and H₂ NOC--CH₂ CO-- in combination with apharmaceutically acceptable diluent.
 2. A composition according to claim1 in which the terminal amino group of the A chain (A₁ glycine) iseither free or protected by an acyl or other blocking substituentcontaining no more than three atoms other than hydrogen selected fromthe group consisting of formyl, acetyl, carbamyl, thiocarbamyl, ##EQU7##and the amino group of the B₂₉ amino acid (lysine) is either free orprotected by an acyl or other blocking substituent containing no morethan three atoms other than hydrogen selected from the group consistingof formyl, acetyl, carbamyl, thiocarbamyl, ##EQU8##
 3. A compositionaccording to claim 1 in which the B₁ amino acid is protected by acetylor acetoacetyl.
 4. A composition according to claim 1 in which the B₁amino acid is protected by2,2-dimethyl-3-formyl-L-thiazolidine-4-carbonyl.
 5. A compositionaccording to claim 1 in which at least one of the substituents is acarbamyl group.
 6. A composition according to claim 1 wherein theinsulin is mono-substituted.
 7. A composition according to claim 1wherein the insulin is di-substituted.
 8. A composition according toclaim 1 wherein the insulin is tri-substituted.
 9. A compositionaccording to claim 1 wherein the A₁ and B₁ protective substituents arethe same.
 10. A composition according to claim 1 wherein the insulin isB₁ (phenylalanine)-N-monocarbamyl insulin.
 11. A composition accordingto claim 1 wherein the insulin is A₁ (glycine) B₁(phenylalanine)-N,N'-dicarbamyl insulin.
 12. A composition according toclaim 1 wherein the insulin is A₁ (glycine)B₁ (phenylalanine)B₂₉(lysine)-N,N',N"-tricarbamyl insulin.
 13. A pharmaceutical compositionaccording to claim 1 in injectable form.
 14. A composition according toclaim 8 wherein the substituents are the same.
 15. A pharmaceuticalcomposition useful for treating diabetes in humans which comprises ahypoglycemically effective amount of a mixture of A₁ (glycine), B₁(phenylalanine), B₂₉ (lysine)-N,N',N"-tricarbamyl insulin and A₁(glycine), B₁ (phenylalanine)-N,N'-dicarbamyl insulin in combinationwith a pharmaceutically acceptable diluent.
 16. A method of treatingdiabetes in humans which comprises parenterally administering to suchhuman a hypoglycemically effective amount of a mono-, di- ortri-substituted insulin in which the terminal amino group of the B chain(B₁ phenylalanine) is protected by an acyl or other blocking substituentcontaining up to 7 carbon atoms selected from the group consisting offormyl, acetyl, trifluoroacetyl, cyclopropane-carbonyl, aceto-acetyl,benzoyl, 2,2-dimethyl-3-formyl-L-thiazolidine-4-carbonyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU9## HOCH₂ CH₂CO--, HOOCCH₂ CO-- and H₂ NOCCH₂ CO--, the terminal amino group of the Achain (A₁ glycine) is either free or protected by an acyl or otherblocking substituent containing no more than 4 carbon atoms and no freeprimary amino group selected from the group consisting of formyl,acetyl, trifluoroacetyl, cyclopropane-carbonyl, acetoacetyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU10## HOCH₂ CH₂CO--, HOOCCH₂ CO-- and H₂ NOC--CH₂ CO-- and the amino group of the B₂₉amino acid (lysine) is either free or protected by an acyl or otherblocking substituent containing no more than 4 carbon atoms and no freeprimary amino group selected from the group consisting of formyl,acetyl, trifluoroacetyl, cyclopropanecarbonyl, acetoacetyl, carbamyl,methylcarbamyl, thiocarbamyl, methylthiocarbamyl, ##EQU11## HOCH₂ CH₂CO--, HOOC--CH₂ --CO-- and H₂ NOC--CH₂ CO--.
 17. A method according toclaim 16 in which the terminal amino group of the A chain (A₁ glycine)is either free or protected by an acyl or other blocking substituentcontaining no more than three atoms other than hydrogen selected fromthe group consisting of formyl, acetyl, carbamyl, thiocarbamyl,##EQU12## and the amino group of the B₂₉ amino acid (lysine) is eitherfree or protected by an acyl or other blocking substituent containing nomore than three atoms other than hydrogen selected from the groupconsisting of formyl, acetyl, carbamyl, thiocarbamyl, ##EQU13##
 18. Amethod according to claim 16 in which the B₁ amino acid is protected byacetyl or acetoacetyl.
 19. A method according to claim 16 in which theB₁ amino acid is protected by2,2-dimethyl-3-formyl-L-thiazolidine-4-carbonyl.
 20. A method accordingto claim 16 in which at least one of the substituents is a carbamylgroup.
 21. A method according to claim 16 wherein the insulin ismono-substituted.
 22. A method according to claim 16 wherein the insulinis di-substituted.
 23. A method according to claim 16 wherein theinsulin is tri-substituted.
 24. A method according to claim 17 whereinthe A₁ and B₁ protective substituents are the same.
 25. A methodaccording to claim 16 wherein the insulin is B₁(phenylalanine-N-monocarbamyl insulin.
 26. A method according to claim16 wherein the insulin is A₁ (glycine)B₁ (phenylalanine)-N,N'-dicarbamylinsulin.
 27. A method according to claim 16 wherein the insulin is A₁(glycine)B₁ (phenylalanine)B₂₉ (lysine)-N,N',N"-tricarbamyl insulin. 28.A method according to claim 13 wherein the substituents are the same.29. A method of treating diabetes in humans which comprises parenterallyadministering to such human a hypoglycemically effective amount of amixture of A₁ (glycine), B₁ (phenylalanine), B₂₉(lysine)-N,N'N"-tricarbamyl insulin and A₁ (glycine), B₁(phenylalanine)-N,N'-dicarbamyl insulin.